A very large number of HIV mutants have been recorded in the Los Alamos HIV Molecular Immunology Database. Interestingly, common mutations, i.e., found in 30-40% of viral isolates, represent few different sequences, while numerous different rare mutations are found only in single or very few individuals. One interpretation of these facts may be that the common mutants represent weak immunogenic sequence variations that have most successfully evaded the selective pressure of CTL recognition, and that more immunogenic rare mutants have not. Furthermore, the principal basis for enhanced immunogenicity of the rarer CTL epitope mutants may be their increased content of non-conservative amino acid substitutions. We propose to explore the hypothesis that the rarer mutant CTL epitopes of HIV are more immunogenic than the common ones, and to establish the proof-of-principal that they may be more effective as vaccines in generating anti-HIV CTL responses. To validate this approach we have selected three HLA-A2 restricted epitopes [Gag p17 77-85; Gag p24 19-27; RT 309-317] since the cellular immune responses to HLA-A2 CTL epitopes are by far the most well studied and the HLA-A2 superfamily has been associated with natural resistance to infection. In a first step, we will synthesize all mutants listed in the Los Alamos Database for these three epitopes (a total of 107 individual peptides). Immunogenicity will be assessed in Aim 2 using HLA-A2 transgenic mouse model. Thus, animals will be immunized, CTL lines generated, and the avidity of these lines will be assessed (1) by ELISpot on: immunizing peptide, the native ligand, and/or common mutants; (2) by intracellular staining for the responding cells; and (3) on T2 target cells pulsed with the priming peptide, the native ligand, and common mutants. The essence of these studies is to demonstrate that these peptide mutants are able to generate specific CTLs that recognize the immunizing peptide, but more importantly, they also recognize the corresponding natural HIV-1 epitope and common mutants. Finally, the breadth of CTL responses induced by a "cocktail" mixture of the most immunogenic peptide mutants identified will be determined using the same model system (Aim 3). Following the proposed proof-of-principle studies, we anticipate having available a series of peptide candidates for toxicity and efficacy in phase I/II clinical trials as part of a phase II SBIR effort. In addition, phase II will include the identification of multiple other CTL epitope mutants for A2 and other common MHC class I haplotypes for the design of a more complete vaccine covering multiple HIV epitopes. [unreadable] [unreadable]